The Bowman-Birk protease inhibitor (BBI) was first described decades ago (Bowman et al., Arch. Biochem. Biophys. 16:109-113 (1948); Bowman et al., Proc. Soc. Exp. Biol. Med. 57:139-140 (1944)). The BBI protein consists of 71 amino acid residues and 7 disulfide bonds, and it has a molecular weight of 7975 daltons (Odani et al., J. Biochem. 74:697-715 (1973)). BBI contains two functional protease inhibitor domains of different specificities. It inhibits both trypsin and chymotrypsin-like proteases (Birk et al., J. Peptide Protein Res. 25:113-134 (1985)), wherein one domain inhibits chymotrypsin-like proteases, and the other inhibits trypsin-like proteases. Chymase and tryptase are serine proteases, which are stored in the cytosol, from which they may be released upon stimulation by potentially pro-inflammatory cells, such as mast cells or macrophages.
The potent ability of certain serine protease inhibitors, such as BBI, to prevent the malignant transformation of cells was discovered in the laboratory of Dr. Ann Kennedy more than two decades ago (e.g., U.S. Pat. Nos. 5,217,717 and 5,338,547; Kennedy et al., Nature 26:825-826 (1978); Yavelow et al., Proc. Nat'l. Acad. Sci. USA 82:5395-5399 (1985); Kennedy, In Protease-Inhibitors as Cancer Chemopreventive Agents, Troll, W, Kennedy, A R (eds), New York, Plenum Press, 1993A, pp. 9-64; Kennedy, Pharmacological Therapeutics 78:167-209 (1998)).
Bowman Birk Inhibitor Concentrate (BBIC) is a soybean-derived extract enriched in the protease inhibitor, BBI, developed by Dr. Kennedy as a cancer chemopreventative agent (Kennedy et al., Nutr. Cancer 19:281-302 (1993B); U.S. Pat. Nos. 4,793,996; 5,217,717 and 5,338,547). The use of BBIC is preferred over crude soybean extract because: a) a very large amount of crude soybean extract would be required to contain amounts of BBI equivalent to the proposed dose of BBIC (approximately 2 quarts of soybean milk or the equivalent amount of tofu); b) crude soybean extract may also contain components which actually counter some of the anticipated beneficial effects of BBI.
Because the anti-carcinogenic activity of BBI is associated with the chymotrypsin-inhibitory domain of BBI (Yavelow et al., 1985), BBIC is quantitated in chymotrypsin inhibitor (CI) units (one CI unit is defined as the amount required to inhibit one mg of chymotrypsin (Kennedy et al., 1993B). In April 1992, BBIC was granted Investigational New Drug status (IND # 34671) from the Food and Drug Administration (FDA), and human trials to evaluate BBIC in several disease states are completed or in progress.
It is clear from animal studies that orally ingested BBI is absorbed and has systemic effects (reviewed in Kennedy, 1998). The structure of the BBI molecule is extremely stable (Birk, 1985; Birk, Meth. Enzymol. 45:695-751 (1975)), such that it survives the digestive process as an intact protease inhibitor capable of inhibiting proteolytic activities. This contrasts with a number of other protease inhibitors which do not survive the digestive process (Kennedy, unpublished). Approximately 50% of the ingested BBI is absorbed into the bloodstream.
BBIC is reportedly a better inhibitor of human chymases than any physiologic protease inhibitor described to date. In a recently finished Phase IIa oral cancer chemoprevention trial in patients with pre-malignant lesions known as oral leukoplakia, daily doses of BBIC led to a significant decrease in lesion size in a dose-dependent manner (Meyskens et al., Proc. Amer. Assoc. Cancer Res. 40:Abstract #2855 (1999).
Significantly, no toxicity has been observed due to BBIC in any human trial (see Table in Detailed Description). No antibodies against BBIC have been found in the sera of any patients receiving BBIC orally (Maki et al., Nutr. Cancer 22:185-193 (1994); Kennedy, personal communication; U.S. Pat. No. 5,961,980).
Absorbed BBI is measurable using antibodies to reduced BBI, produced by injection into experimental animals and utilized in immunoassays (Wan et al., 1995). BBI has been assessed in the blood, tissue and urine of rodents and dogs after the ingestion of BBIC permitting pharmacokinetic studies, although it has not yet been feasible to measure BBI levels in the blood of humans after oral BBIC dosing. However, it has been found in the urine, starting within several hours after a single oral dose (Wan et al., Cancer Epidem. Biomarkers & Prevention 8:601-608 (1999)). Of note, studies in orally-dosed animals have shown that some BBI can be subsequently found in the CNS even when the blood-brain barrier is intact (Kennedy, A R, personal communication).
BBIC has also proven in many instances to be an effective anti-inflammatory agent. BBIC has been demonstrated to have a suppressive effect on inflammation occurring in carcinogen-treated rodents rodents, as measured by the level of inflammatory infiltrates or lymphoid aggregates, in organs such as the colon and esophagus. For example, in the treatment of ulcerative colitis, an inflammatory bowel disease (Ware et al., Digestive Diseases and Sciences 44:896-90 (1999)), inflammation was significantly reduced following treatment with BBIC. Moreover, the chemical induction of ulcerative colitis in rats resulted in the induction of many proteolytic activities in the lesioned, inflamed tissues, on which BBI/BBIC reportedly showed a highly significant inhibitory effect on essentially all of the induced proteolytic activities (Hawkins et al., Digestive Diseases and Sciences 42:1969-1980 (1996)). In addition, BBI/BBIC treatment resulted in a suppression of cancer development and a reduction in the levels of inflammation, as measured by the level of inflammatory infiltrates or lymphoid aggregates in the colon (Kennedy et al., 1993A).
Several possible mechanisms by which BBIC may cause these effects have been proposed. First, it has been suggested that BBIC interferes with the inflammatory response by reducing the production of oxygen radicals in in polymorphonuclear leukocytes. Second, BBIC reportedly decreases interleukin-1 (IL-1) release, which is a well known, pro-inflammatory cytokine, participating in a wide variety of immune and inflammatory reactions. Third, BBIC has been shown to have the ability to inhibit the malignant transformation of cells; it has been hypothesized that BBI may inhibit cell transformation by affecting the function of certain oncogenes/proto-oncogenes (e.g., c-myc and c-fos). Nevertheless, little is known yet about either the cellular or molecular mechanisms by which BBIC can modulate or ameliorate autoimmune diseases.
BBI has been shown to efficiently inhibit several identified proteases released from human inflammation-mediating cells. These include human leukocyte elastase Tikhonova et al., Biochemistry (Moscow) 59:1295-1299 (1994); Larionova et al., Biochemistry (Moscow) 58:1437-44 (1993)) and human cathepsin G (Larionova et al., 1993; Gladysheva et al., Biochemistry (Moscow) 59(4):513-518 (1994)), which can efficiently destroy matrix molecules and severely damage tissues.
It is also known that BBI, as well as several other inhibitors of chymotrypsin proteolytic activity, have the ability to prevent the induction of superoxide anion radicals and hydrogen peroxide from stimulated human polymorphonuclear leukocytes and macrophage-like cells (Frenkel et al., Carcinogenesis 8:1207-1212 (1987); Ware et al., Nutr. Canc. 33:174-177 (1999)). Proteases and free radicals produced by macrophages are closely associated with the production of inflammation. For example, Multiple Sclerosis (MS) is characterized by inflammation and increased numbers of activated immunocytes of macrophage and T cell lineage (Hauser et al., In Harrison's Principles of Internal Medicine. Fauci et al. (eds). New York, McGraw-Hill, 1998, pp. 2409-2419).
Multiple Sclerosis (MS).
Multiple sclerosis (MS) is a neuroinflammatory disease of the central nervous system (CNS) characterized by chronic inflammation, demyelination and gliosis. Pathologically, MS is characterized by well-demarcated, macroscopic lesions, called plaques, in the brain white matter and, less frequently, gray matter. Acute lesions are characterized by perivenular cuffing and infiltration of T lymphocytes and macrophages, along with a few B cells and plasma cells. MS is reportedly an autoimmune disorder, likely triggered by environmental exposure in a genetically susceptible host. Complications from MS may affect multiple physiological systems and require profound changes in lifestyle for patients and their families. MS affects 350,000 Americans and is the second most frequent cause (after trauma) of neurologic disability in early to middle adulthood (Hauser et al., 1998).
MS is a complex disease, manifested in progressive or relapsing modalities, or combinations thereof. Proteolysis of myelin appears key to demyelination, which, in association with perivenular inflammation, is a major pathological feature in multiple sclerosis. Much of the investigative interest in MS has focused on the possible toxic effects on CNS myelin of locally accumulated lymphocytes and their components/products (Bar-Or et al., J. Neuroimmunol. 100:252-259 (1999); Wucherpfennig et al., J. Clin. Invest. 100(5):1114-1122 (1997).
Proteases are associated with many facets of immune system function and immune system disorders (Cuzner et al., J. Neuroimmunol. 6:1-14 (1999); Vaday et al., J. Leukoc. Biol. 67:149-159 (2000)). A variety of proteases are increased in MS lesions, including lysosomal proteases and matrix metalloproteinases gelatinase A and B (MMP-2 and 9, respectively) (Cuzner et al., 1999; Halonen et al., J. Neurol. Sci. 79:267-274 (1987); Kieseier et al., Curr. Opin. Neurol. 12:323-336 (1999); Hartung et al., J. Neuroimmunol. 107:140-147 (2000); Bever et al., Neurology 53:1380-1381 (1999); Maeda et al., J. Neuropathol. Experimental Neurol. 55:300-309 (1996)).
Macrophages are also observed in association with chronic MS plaques (Hauser et al., 1998), even in early plaques, raising the possibility the macrophages may release myelinotoxic agents as well as serving a scavenger role (Cuzner et al., 1999; Raine, Ann. Neurol. 36:S61-72 (1994)). Macrophages are known to release MMPs, which are characteristically released from cells in the form of an inactive proenzyme, which must be activated by proteolytic removal of a propeptide (Atkinson et al., Biochem. J. 288:605-611 (1992); Cuzner et al., 1999; Hartung et al., 2000). This activation step can be carried out by other MMPs (Atkinson et al, 1992), or by serine proteases, such as plasmin, cathepsin G, chymase and trypsin (Cuzner et al., 1999; Hartung et al., 2000; Murphy et al., Ann. N.Y. Acad. Sci. 667:1-12 (1992); Brosnan et al., Nature 285:235-237 (1980)).
Mast cells also frequently accumulate in the cellular areas of MS plaques (Cuzner et al., 1999: Ibrahim et al., J. Neuroimmunol. 70:131-138 (1996)). Therefore, it is of interest that mast cells reportedly contain two serine proteases (cathepsin G [Ki=1.2 nM] and chymase [Ki=50 pM]), which are released with histamine upon degranulation. Active at neutral pH, the serine proteases may not only play an important initial role in the activation of MMPs in the pro-inflammatory enzymatic cascade, but they could account for the cleavage of the myelin components and release of the encephalitogenic fragments (Opdenakker et al., Immunol. Today 15:103-107 (1994)). Taken together, these observations indicate that mast cell proteases play a role in MS, and suggest that inhibition of serine protease activity is a potentially important therapeutic approach in MS, particularly if the production of naturally occurring anti-proteinases is impaired in MS lesional areas. Currently, BBI is the best known natural inhibitor of mast cell chymase (Ware et al., Arch. Biochem. Biophys. 344:133-138 (1997)).
Agents presently used prophylactically against MS relapses include interferon β1a, β1b, and copolymer-1. These agents are administered by subcutaneous or intramuscular injection on a daily or every other day basis. While generally well tolerated, the benefit of these therapies is limited to reducing the MS relapse rate by only about one third, when compared to placebo recipients. Moreover, neutralizing antibodies against interferon are produced within 12 months of initial treatment by significant numbers (20-40%) of those patients receiving current therapies, causing those patients to return to their pretreatment relapse rate. Acute relapses may be treated with a brief course of intravenous methylprednisolone, followed by oral prednisone. However, such short-term glucocorticoid therapy is associated with fluid retention, weight gain, gastric disturbances and emotional lability, which may require further treatment.
Chronically progressing MS is sometimes treated with immunosuppressants, such as methotrexate, azathioprine or cladribine. However, while these agents are of modest efficacy, regular monitoring of a patient's blood cell counts and liver functions during therapy is advised due to potential toxicities. Spasticity can be treated with agents, such as baclofen, diazapam, or clonazepam, but these are all of limited efficacy and can be counterproductive for patients who require a degree of rigidity for daily activities, such as walking.
Experimental Autoimmune Encephalomyelitis (EAE).
Experimental autoimmune encephalomyelitis (EAE) is a long-established disease model for MS. First described in monkeys (Rivers et al., J. Exp. Med. 58:39-53 (1933), this paradigm has been reproduced in several species, including mice and rats. EAE is induced by immunizing with myelin components, purified myelin proteins, or by peptide fragments resulting from the cleavage of stable encephalitogenic peptides from myelin, using a protease released from degranulated mast cells at neutral pH (Dietsch et al., Cell. Immunol. 135:541-548 (1991); Constantinescu et al., Immunologic Res. 17:217-227 (1998); Constantinescu et al., J. Immunol. 161:5097-5104 (1998)). EAE can also be induced ‘passively’ by adoptive transfer of antigen-reactive T helper cells from an immunized animal.
Histopathologically, EAE is characterized by CNS inflammation with macrophage and lymphocytic infiltrates and varying degrees of demyelination (Raine, In Textbook of Neuropathology, Davis R L (ed) Baltimore, Md., Williams and Wilkins, 1990, pp. 356-358). The disease manifests clinically with paralysis, beginning at the tail and spreading rostrally to the hindlimbs and forelimbs, and in advanced stages affects breathing and causes death.
Experimental Autoimmune Neuritis (EAN).
Oral BBIC administration has also demonstrated a profound inhibitory effect in rats with experimental autoimmune neuritis (EAN), an animal model of autoimmune peripheral nerve demyelinating disease having clinical, pathological and electrophysiological similarities to human Guillain-Barre Syndrome (GBS). GBS is an autoimmune, neuroinflammatory disorder related to MS, but affecting primarily the peripheral nervous system (PNS) (Shang et al., J Immunol. 160:467 (1998); Owens et al., Immunol. Today 15:566 (1994)).
EAN is a T cell mediated disease that can be transferred by CD4-positive antigen-specific Th1 cells. Histopathologically, EAN is characterized by T cell and macrophage infiltration of the nerve roots (Weber et al., J. Cell. Biol. 134:1063 (1996)), demyelination and axonal injury (Waksman et al., J. Exp. Med. 102:213-236 (1955); Rosen et al., Muscle and Nerve 13:629-636 (1990); Vaddi et al., J. Immunol. 153:4721 (1994)). The infiltration of cells to the target tissue is accompanied with increased expression of adhesion molecules, pro-inflammatory cytokines and chemokines, both by the infiltrating cells and at the site of the immunological insult. Clinically, EAN manifests in rats first with tail paralysis, and progresses rostrally with paralysis of the hindlimbs, then the forelimbs. However, when model animals were treated with BBIC, a reduction was seen in both the extent of demyelination and the accumulation of inflammatory cells in the PNS tissue, comparable to the effect in EAE.
EAN is induced in rats by the injection of whole peripheral nerve tissues (Waksman et al., 1955; Rostami, Springer Semin. Immunopathol. 17:29-42 (1995)), a protein fraction (P0 or P2) isolated from peripheral nerve myelin (Brostoff et al., Nat. New Biol. 235:210-212 (1972); Rostami et al., Ann. Neurol. 16:680-685 (1984)), or by synthetic peptides corresponding to the myelin proteins (peptide SP26 corresponds to the 53-78 amino-acid sequence of the myelin P2 protein) (Rostami et al., J. Neuroimmunol. 30:145-151 (1990).
Inflammatory autoimmune diseases, such as rheumatoid arthritis, and neuro-inflammatory autoimmune diseases, such as Multiple Sclerosis (MS) and Guillain Barre Syndrome, exert a major impact on the health of the American population. MS is particularly devastating because of the extensive morbidity and premature fatalities in relatively young, productive individuals afflicted with the disease. Current therapeutic approaches to MS, such as treatment with glatiramer or beta-interferon (INF-β) have resulted in only relatively modest benefits. However, the disease is not well controlled. The use of other therapeutic agents, such as corticosteroids and immunosuppressive agents, in recent years has also been limited by inconsistent benefits and/or cumulative toxic effects. Therefore, from both a social and a medical standpoint, there is a major need for a reliable and effective non-toxic method for treating the chronic inflammatory effects that play pathogenic roles in MS, GBS, and other autoimmune diseases.